Summary Chemotactic cell migration underlies embryonic development, wound healing and immune responses. Furthermore, aberrant chemotaxis leads to chronic inflammatory disease and tumor metastasis. My laboratory has long been interested in signaling mechanisms that control cell behaviors in chemotaxis. In a chemical gradient, cells polarize intracellular signaling and migrate toward chemoattractants. Ligand binding to chemoattractant receptors selectively activates two kinases, TORC2 and PI3K, at the front of cells. TORC2 and PI3K are the master kinases that induce cytoskeletal remodeling to extend pseudopods and function immediately downstream of chemotactic receptors. Polarized activation of these two kinases is essential for creating the leading edge of cells that migrate in a chemical gradient. Despite the critical relevance to basic and medical science, an understanding of the spatial and temporal regulation of TORC2 and PI3K remains incomplete. An overarching challenge is to decipher how the TORC2 and PI3K signaling pathways are regulated at the front versus the back of cells in a chemical gradient. To explore mechanisms that regulate the TORC2 pathway, we will take advantage of our recently developed biochemical systems. In these systems, chemoattractant-regulated activation and inhibition of TORC2 are faithfully reconstituted with purified TORC2 and two small GTPases, Rho and Ras. These new assays will identify the biochemical and biophysical mechanisms that create distinct chemotactic signaling at the leading and trailing edges of migrating cells. The mechanistic principle that is determined will be tested and translated in our cellular reconstitution systems using knockout cell lines expressing WT and mutant TORC2 and its regulatory components. Using live-cell imaging with FRET microscopy and single-molecule microscopy, we will place the signaling principle in a spatial and temporal context in migrating cells. For the PI3K pathway, we will analyze proteins that control the localization of the PIP3 phosphatase PTEN to the plasma membrane at the back of cells. The rear localization of PTEN enables PIP3 signaling activation at the leading edge and its inhibition at the trailing edge. We will also determine the function of the identified mechanisms that control PTEN localization in tumorigenesis and metastasis in mouse xenograft models expressing engineered PTEN molecules with altered localization. These studies will elucidate the fundamental logics by which polarization of intracellular signaling is established in cells during chemotactic migration and the physiological importance of this signaling in vivo.